The innate immune system serves is the first response to diseased cells and infectious agents. Natural Killer (NK) cells and macrophages are primary components of this system. Although their functions differ, they both require the expression of NKLAM (Natural Killer Lytic-Associated Molecule) for optimal function. Accordingly, elucidating the role of NKLAM in innate immunity has important biological and clinical significance. NKLAM is up-regulated in NK cells upon exposure to tumor cells or cytokines that activate cytolytic function. NKLAM is up-regulated in macrophages upon exposure to bacteria or pro-inflammatory cytokines. We generated NKLAM-deficient (KO) mice; they have less NK activity than WT (wild type) mice. They exhibit greater tumor growth, tumor dissemination and metastasis than WT mice. NKLAM KO mice also have defects in macrophage anti-bacterial function in vivo and in vitro. NKLAM is a member of a small, highly conserved, unique group of RING-in between-RING (RBR) E3 ubiquitin ligases. RBR E3 ubiquitin ligases play a key role in cellular physiology and are involved in the pathogenesis of many diseases, including cancer, autoimmune diseases and neurological disorders. There is great interest in drug discovery to target them. We identified key potential substrates of NKLAM-mediated ubiquitination, including the transcription factors STAT1 and c-myc, Bcl-2 and UCKL-1 (uridine cytidine kinase like-1), a protein that promotes tumor growth. The central nature of these substrates in cell signaling, growth and survival suggests that drug modulation of NKLAM has significant potential for treating cancer and infectious diseases. We found that activated NK cells releases exosomes containing NKLAM. NKLAM+ exosomes promote tumor cell death in vitro. Preliminary data indicate that NKLAM+ exosomes enter target cells and deliver NKLAM. Important unanswered questions that will be addressed are the effect of NKLAM on critical substrates in effector cells and in target cells. The role of NKLAM in macrophages is undoubtedly different from its role in NK cells in that killing of bacteria occurs intracellularly. Our ongoing studies suggest that NKLAM modulates the signaling events that activate the macrophage anti-bacterial program. A key regulator of this pathway is STAT1. We will test the hypothesis that the E3 ubiquitin ligase activity of NKLAM plays a role in the anti-tumor and anti-microbial functions of NK cells and macrophages with the following three interconnected but independent aims: Aim 1: Elucidate the role of NKLAM in exosome-mediated killing of tumor cells. We will test the hypothesis that upon entry of NK-derived exosomes containing NKLAM into tumor cells, NKLAM interacts with critical substrates, resulting in cell death. We will test the ability of exosomes from NKLAM WT, NKLAM KO and NKLAM ligase defective NK cells to kill tumor cells in vivo using a mouse model of multiple myeloma, a disease increasing in frequency, especially in veterans. Aim 2: Determine how NKLAM affects the anti-bacterial function of macrophages in vitro and in vivo. We will infect NKLAM KO and WT mice with S. pneumoniae and S. aureus, pathogens that are leading causes of disease. In light of increasing antibiotic resistance, the importance of these in vivo studies is magnified. Aim 3: Identify the mechanism, sites and types of ubiquitination mediated by NKLAM. We will examine substrates important in tumor growth and anti-bacterial killing. This will provide a better understanding of the role of NKLAM in anti-tumor and anti-bacterial immunity and reveal key regulatory steps susceptible to therapeutic interventions for diseases that afflict our veteran population.